This R01 application responds to PAR-10-190: Vulvodynia - Systematic Epidemiologic, Etiologic or Therapeutic Studies. Our long-term goal is to develop an understanding of the vulvodynia pain mechanism leading to a mechanism-based disease classification and ultimately to a mechanism-based therapy. Our research team has reported a mechanistic connection between yeast products, regional fibroblast activation, pro-opiomelanocortins, and localized provoked vulvodynia (LPV). Fibroblasts are now recognized as more than structural cells as they not only respond to signals but can prodigiously produce many different biologic mediators, including those that promote pain. Fibroblasts also exhibit considerable regional specialization. We discovered that fibroblasts from the vulvar vestibule produce markedly elevated levels of pro-inflammatory, pro-pain mediators following activation with yeast cell wall products. In particular, heightened pro-inflammatory mediator responses are generated by fibroblasts from the vulvar vestibule of LPV-afflicted women. This may be related to single nucleotide polymorphisms (SNP) in the melanocortin-1 receptor (MC1R) that enhance inflammatory mediator production. We propose that the vulvar vestibule of all women possesses a unique inflammatory/pain-inducing responsiveness and that vulvodynia pain reflects an extreme but natural inflammatory phenomenon. We hypothesize that vulvodynia arises 1) in a region of the genital tract predisposed to inflammation, 2) in the presence of specific irritants such as yeast, that are 3) exacerbated by genetic predisposition. To significantly advance and impact the field, we have assembled a multidisciplinary team, experienced in LPV, fibroblast biology, and inflammation to achieve the following three aims. Specific Aim 1: To determine whether pro-inflammatory fibroblasts segregate to painful areas of the vulva. Using lower genital tract pain mapping, we will discover whether pro-inflammatory fibroblasts localize to painful anatomic regions in situ. Fibroblast strains will be developed from painful and non-painful areas of the vulva and their biosynthetic capabilities for pro-inflammatory and other mediators determined after exposure to key fibroblast activating cytokines. Specific Aim 2: To determine whether yeast or yeast products activate fibroblasts via Toll-like receptors (TLR) and whether specific MC1R SNPs modify that response. We will determine whether the LPV-afflicted patients carry a different pattern of yeast species and yeast load, and whether yeast cell wall products initiate, through toll-like receptors, a pro-inflammatory, pain-inducing response from fibroblasts derived from painful regions. Specific Aim 3: To determine whether pro-opiomelanocortin loss-of-function promotes vulvodynia. We will investigate whether loss-of-function melanocortin-1 receptor SNPs enhance site-specific fibroblast activation, and can be identified with a simple clinical measure, skin colorimetry. We will assess an anti-inflammatory melanocortin derivative with therapeutic potential for vulvodynia and investigate the underlying molecular mechanism(s).